Generation of functional human serotonergic neurons from fibroblasts

Abstract

The brain's serotonergic system centrally regulates several physiological processes and its dysfunction has been implicated in the pathophysiology of several neuropsychiatric disorders. While in the past our understanding of serotonergic neurotransmission has come mainly from mouse models, the development of pluripotent stem cell and induced fibroblast-to-neuron (iN) transdifferentiation technologies has revolutionized our ability to generate human neurons in vitro. Utilizing these techniques and a novel lentiviral reporter for serotonergic neurons, we identified and overexpressed key transcription factors to successfully generate human serotonergic neurons. We found that overexpressing the transcription factors NKX2.2, FEV, GATA2 and LMX1B in combination with ASCL1 and NGN2 directly and efficiently generated serotonergic neurons from human fibroblasts. Induced serotonergic neurons (iSNs) showed increased expression of specific serotonergic genes that are known to be expressed in raphe nuclei. iSNs displayed spontaneous action potentials, released serotonin in vitro and functionally responded to selective serotonin reuptake inhibitors (SSRIs). Here, we demonstrate the efficient generation of functional human serotonergic neurons from human fibroblasts as a novel tool for studying human serotonergic neurotransmission in health and disease.

Evaluating cell reprogramming, differentiation and conversion technologies in neuroscience

“…c | To facilitate neurotransmitter-specific iN conversion, cocktails of specific transcription factors can be added to shape a specific neuronal identity. These lineage-specifying transcription factors are typically well known for their essential roles during the development of the targeted neuronal subtype in vivo 88,89,99,119,122–124,196 . d | Manipulation of signal transduction pathways through growth factors and small molecules that inhibit the transforming growth factor-β (TGFβ)–ALK–SMAD pathway and glycogen synthase kinase 3β (GSK3β), as well as the promotion of cyclic AMP signalling, increases iN conversion efficiencies.…”

“…It is thus of crucial importance for future studies to build neuronal profiles based on multimodal analyses, including broad molecular signatures and deep electrophysiological properties. For example, bulk and single-cell transcriptomic technologies provide an in-depth molecular signature that can be directly compared to the human brain transcriptome database 99 . For functional evaluation, patch clamping remains the gold standard but only permits us to assess a handful of selected neurons within several thousands of cells.…”

Induced dopaminergic neurons: A new promise for Parkinson’s disease

“…This method has been successfully extended to the human system, where fetal and postnatal human fibroblasts are converted to iN with BAM plus NeuroD1 [11]. Many groups have successfully converted mouse, porcine, non-human primate [12] or human fibroblasts into iN, DA neuronal progenitors (iDPs) [13], [14], dopaminergic neurons [8], [15], [16], motor neurons (hiMNs) [17], [18], [19], peripheral sensory neurons [20], striatal medium spiny neurons (MSNs) [21], [22], cholinergic neurons [23], serotonergic neurons [24], [25], GABAergic neurons [26], glutamatergic neurons [27], neural precursor cells [28] and oligodendrocyte progenitor cells [29], [30], with different combinations of transcription factors and media additives (small molecule compounds and growth factors).…”

Advancing drug discovery for neuropsychiatric disorders using patient-specific stem cell models

“…Certainly assays relying on longer time in culture are likely to better capture features present only in mature neurons, but at the cost of diminished feasibility and scalability: even with modern culture techniques and robotics, maintaining neurons in culture for 8 weeks or more can be labor intensive. In part for this reason, a parallel line of work is investigating ‘shortcuts’ for rapid generation of neural cells without requiring iPSC culture, so-called induced neurons, has promise and has begun to be used to reveal disease phenotypes 110,111 .…”